摘要目的：饲料氨基酸调节乳蛋白合成的机制还不清楚。大量的研究表明，氨基酸的信号是通过哺乳动物雷帕霉素靶蛋白（mTOR）通路传递的，但细胞外氨基酸激活哺乳动物雷帕霉素靶蛋白复合物1的机制还不清楚。 我们探讨了T1R1/T1R3异二聚物作为氨基酸的直接感受器,通过感知氨基酸,进一步通过mTOR信号通路影响乳腺上皮细胞上牛乳蛋白质的合成可能性。

方法：通过T1R1 siRNA抑制小鼠乳腺上皮细胞系(HC11)T1R1的表达。 使用免疫印迹方法检测mTOR信号通路和β-酪蛋白的表达，使用实时定量RT-PCR的方法检测氨基酸转运体mRNA数量的变化。76468

结果:在HC11细胞和小鼠乳腺组织中检测到T1R1和T1R3的mRNA与蛋白。T1R1的 siRNA抑制HC11细胞中β-酪蛋白的合成。mTOR、核糖体S6蛋白激酶和4EBP1的磷酸化在T1R1基因沉默 HC11细胞下降到25%，50%和30%，表明T1R1基因沉默抑制mTOR信号通路的活性。T1R1基因沉默增加三种重要的氨基酸转运子（SLC1A5、SLC3A2和SLC7A5)mRNA丰度。mTOR信号通路的活化被T1R1 siRNA、SLC7A5和SLC3A2抑制剂部分抑制（BCH，10 mM），并且结合了这两种抑制作用，有累加效应。

结论:T1R1/T1R3作为小鼠乳腺上皮细胞外氨基酸的传感器，参与了乳蛋白合成的调控。

毕业论文关键词: T1R1·T1R3·氨基酸传感·mTOR

T1R1 HC11 cells function analysis

Abstract:Purpose The mechanism of dietary amino acids in regulating milk protein synthesis at the translational level is not well understood。 Numerous studies have shown that the amino acid signal is transferred through the mammalian target of rapamycin (mTOR) pathway, however the extracellular amino acid-sensing mechanism that activates mTOR complex 1 is unknown。 We tested the hypotheses that the T1R1/T1R3 heterodimer functions as a direct sensor of the fed state and amino acid availability preceding the mTOR pathway, and affects milk protein synthesis in mammary epithelial cells。 

Methods The expression of T1R1 was repressed by T1R1 siRNA in mouse mammary epithelial cells model (HC11)。 Western blot was used to analyze activity of the mTOR pathway and β-casein expression, and quantitative real-time RT-PCR was used to analyze the change in mRNA abundance of amino acid transporters。 

Results The transcripts and proteins of T1R1 and T1R3 were detected in HC11 cells and mouse mammary gland tissue。 siRNA silencing of T1R1 repressed β-casein synthesis in HC11 cells both  with and without essential amino acids present in the culture medium。 The phosphorylation of mTOR, S6K and 4EBP1 in T1R1 knockdown HC11 cells declined to 25%, 50% and 30%, indicating T1R1 knockdown repressed the activity of the mTOR pathway。 T1R1 knockdown increased the mRNAs coding three important amino acid transporters (SLC1A5 and SLC3A2/SLC7A5)。 Activation of the mTOR pathway was partially repressed by T1R1 siRNA or SLC7A5/SLC3A2 inhibitor (BCH, 10mM), and the combination of these two treatments further repressed the activity of this pathway。

Conclusion T1R1/T1R3 serves as sensor of extracellular amino acids in mouse mammary epithelial cells, and involed in milk protein synthesis regulation。

目录

1、前言 1

2、材料与方法 2

2。1、材料 2

2。2、方法 2

2。2。1、细胞培养 2

2。2。2、RNA干扰 3

2。2。3、抑制性处理 3

2。2。4、实时定量RT-PCR 3

2。2。5、蛋白质印迹分析